Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars

The objective of this study was to evaluate the effects of 6-benzylaminopurine (BAP) and α‐naphthaleneacetic acid (NAA) combinations, basal media and beta‐lactam antibiotics on in vitro organogenesis from mature stem segments of 'Pêra', 'Valência' and 'Bahia' sweet oranges and 'Cravo' rangpur lime. For induction of shoot regeneration, the segments of the four cultivars were placed on Murashige and Skoog (MS) medium containing the following BAP/NAA concentrations: 0.0/0.0; 0.25/0.0; 0.25/0.25; 0.5/0.0; 0.5/0.5; 1.0/0.0; 2.0/0.0; 2.0/0.25; 2.0/0.5; and 2.0/1.0 mg L-1. In order to test the influence of the culture media on shoot-bud induction, (MS), Murashige and Tucker (MT), and woody plant medium (WPM) formulations were evaluated, associated with the best combination of plant growth regulators obtained in the previous experiment. The influence of four beta‐lactam antibiotics (timentin, cefotaxime sodium salt, meropenem trihydrate and augmentin) on shoot regeneration was determined. Better regeneration responses were achieved when internodal segments were cultured onto MS-based medium with 500 mg L-1 cefotaxime with the following BAP/NAA concentrations: 0.5 + 0.25 mg L-1 for 'Cravo', 1.0 + 0.25 mg L-1 for 'Valência' and 'Bahia', and 1.0 + 0.5 mg L-1 for 'Pêra'. Genotype, growth regulators, basal media and beta-lactam antibiotics affect the morphogenetic response in mature tissues of citrus.


Introduction
Several methods for genetic transformation of citrus have been described in the literature, but so far the most effective are those using Agrobacterium-mediated transformation of juvenile materials, such as zygotic embryos, hypocotyls, epicotyls, and cotyledons (Fleming et al., 2000;Al-Bahrany, 2002;Costa et al., 2002;Oliveira et al., 2009).Plants regenerated from these explant sources have long juvenile stages before initial fruit production, and many years are necessary before evaluating the horticultural and commercial traits introduced into the transgenic plants.An ideal protocol for citrus plant transformation should be based on the use of mature tissues as explant sources, circumventing the juvenile phase and permitting the analysis of the introduced traits in a relatively short period of time (Cervera et al., 2000;Peña et al., 2001;Almeida et al., 2003;Cervera et al., 2008;Rodríguez et al., 2008).
For successful genetic transformation of mature tissue, the first step is the establishment of an efficient plant regeneration system, since in vitro culture using mature tissue as explants is still far from routine (Almeida et al., 2003;Rodríguez et al., 2008).Reasons for this include the relatively low responsiveness of woody plants to exogenous growth regulators and the failure of standard surface sterilization techniques (Cervera et al., 2008).A culture medium with optimal mineral supply and combination of plant growth regulators will increase the success for recovery of transgenic citrus plants from transformed cells.Furthermore, the addition of antibiotics to the culture medium may help to eliminate contamination by bacteria, which hinder the in vitro establishment of explants from mature tissues.
Little is known about the effects of cultivars, growth regulators, basal media and beta-lactam antibiotics on in vitro shoot regeneration from mature citrus tissue.Beta-lactam antibiotics, such as carbenicillin and cefotaxime, are the most commonly used antibiotics in plant transformation protocols, since they have a broad spectrum of activity against bacteria and a low toxicity to eukaryotes (Bhau & Wakhlu, 2001;Yu et al., 2001).It has been speculated that some antibiotics may act as regulators of morphogenic development, and that they can enhance the in vitro response (Humara et al., 1999;Costa et al., 2000;Danilova et al., 2004).
The objective of this study was to evaluate the effects of different 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) combinations, basal media and beta-lactam antibiotics on in vitro shoot regeneration from mature stem segments of 'Pêra', 'Valência' and 'Bahia' sweet oranges and 'Cravo' rangpur lime.

Materials and Methods
The experiments were carried out in the plant tissue culture laboratory (Bioagro) at Universidade Federal de Viçosa, Minas Gerais, Brazil, from March to June 2005.Internodal segments, approximately 1.0-1.5-cmlong, from greenhouse-grown adult plants of 'Pêra', 'Valência', and 'Bahia' sweet oranges [Citrus sinensis (L.) Osbeck] and 'Cravo' rangpur lime (Citrus limonia Osbeck) were used as sources of the mature explants.Rejuvenation was applied to facilitate in vitro culture of explants from mature tissue, by grafting buds into juvenile rootstocks.These mother plants were drastically pruned to stimulate the sprouting of the basal buds, which could retain juvenile characters.These plants were regularly sprayed with the fungicide Benlate (Benomyl) at 1% (v/v) to prevent contamination during in vitro culture.Vigorous newly elongated lateral branches, at least 12-inch long, were collected in a semi-hardened stage.Under aseptic conditions, elongated lateral branches were sterilized at the surface using 70% (v/v) ethanol for 2 min, followed by immersion in a 5% (v/v) commercial solution of sodium hypochlorite containing 0.1% (v/v) Tween-20 for 20 min, and sequentially rinsed in sterile distilled water.
In order to test the influence of the culture media on shoot-bud induction, three formulations were evaluated: MS, MT (Murashige & Tuker, 1969) and woody plant medium (WPM) (Lloyd & McCown, 1980).The best combination of plant growth regulators associated with the four genotypes was used in the media formulation: 0.5 mg L -1 BAP + 0.25 mg L -1 NAA for 'Cravo', 1.0 mg L -1 BAP + 0.25 mg L -1 NAA for 'Valência' and 'Bahia', and 1.0 mg L -1 BAP + 0.5 mg L -1 NAA for 'Pêra'.Explants were initially incubated in the dark at 26±2 o C for 30 days and, then, transferred to a 16/8-hour (light/dark) photoperiod, and 36 µmol m -2 s -1 light radiation provided by two 20 W white fluorescent lamps for more 30 days.

Elongated shoots obtained in all cultivars
(1-1.5-cm long) were excised and subcultured on MS medium supplemented with 0.5 mg L -1 NAA for root induction during 60 days.Rooted shoots were transplanted to plastic cups containing sterile soil, sand and vermiculite (1:1:1, v/v/v) and were placed on illuminated shelves under 24 µmol m -2 s -1 irradiance, provided by two 20 W white fluorescent lamps.The plants remained under this condition for 45 days, and then they were transferred to greenhouse for additional 60 days.
For all experiments described above, the cultures were sub-cultured after every 15-20 days on shoot induction media to obtain good growth.After culture for eight weeks, percentage of explants forming shoots and number of shoots per explant were recorded and statistically compared in each factor separately, following a completely randomized design.Twenty shoots were used for plate and five plates per experiment.Each experiment was repeated twice.Data were submitted to analysis of variance, and means were compared by t test or the Tukey-Kramer multiple comparison test at 1% probability.

Results and Discussion
Plant regeneration via direct and indirect organogenesis was obtained on the surface of the cut zone from cultured internodal explants of all four citrus cultivars tested.Small amounts of compact callus appeared on the cut surface within two weeks of culture, in segments incubated in darkness.Shoot buds differentiated from these calluses two weeks after transferring the cultures to the light.Significant differences were observed for cultivars, basal media and antibiotics concentrations, in both percentage of explants forming shoots and average number of shoots per explants (Table 1).All null hypotheses on the effects of interaction between cultivars x BAP/NAA combinations, cultivars x antibiotics concentrations or cultivars x culture media were accepted.The absence of significance in the last interaction indicates that the cultivars did not differ in the various basal medium formulations.
Regeneration increased as the level of BAP in the culture medium increased from 0.25 to 1.0 mg L -1 , irrespective of the citrus cultivar and NAA concentration (Table 2).At 2.0 mg L -1 BAP, with or without NAA, the percentage of explants forming shoots decreased in most of the assessed cultivars.The combination of BAP and NAA was essential for maximum shoot production in all cultivars, although the balance between these Pesq.agropec.bras., Brasília, v.45, n.7, p.654-660, jul.2010 growth regulators for optimal shoot regeneration varied among cultivars.For 'Cravo', the highest number of shoots per explant was obtained in 0.5 mg L -1 BAP + 0.25 mg L -1 NAA, whereas 1.0 mg L -1 BAP + 0.25 mg L -1 NAA was the best combination for 'Valência' and 'Bahia'.'Pêra' produced more shoots per explant in 1.0 mg L -1 BAP + 0.5 mg L -1 NAA.
Plant regeneration of explants from mature citrus tissue has been reported before only for 'Pineapple', 'Pêra', 'Valência', 'Hamlin' and 'Natal' (Cervera et al., 1998;Almeida et al., 2003;Kobayashi et al., 2003).Tissue regeneration had not been reported yet for 'Bahia', the most important edible orange in Brazil and in the USA (Washington navel orange), and 'Cravo' rangpur lime, an important citrus rootstock.
It has previously been reported that, differently from most juvenile explants, explants from mature citrus plants require balanced combinations of BAP and NAA in the culture media for maximum shoot regeneration (Almeida et al., 2003;Rodríguez et al., 2008).The present work corroborates these findings and further demonstrates that the optimal hormone concentrations are specific to cultivar, as it has been previously reported for juvenile explants (Bordón et al., 2000;Costa et al., 2004;Mendes et al., 2008).
Explants cultured in MS and MT media stimulated more morphogenesis than the WPM medium, irrespective of the cultivar analyzed (Table 3); however, WPM induced larger shoots than the MS and MT basal media.Similar observations have been reported for 'Pêra' (Kobayashi et al., 2003) and 'Clementine' mandarin (Cervera et al., 2008) using mature tissue as explant sources.The composition of the culture media used for citrus tissue culture is usually based on the nutrients and vitamins of MS and MT media, although WPM has been successfully used for tissue culture of recalcitrant woody species (Lloyd & McCown, 1980).It has been suggested that the lower Table 2. Effect of 14 combinations of benzylaminopurine (BAP) and naphthalene acetic acid (NAA) concentrations on shoot organogenesis from internodal stem segments of citrus cultivars, after 60 days in MS-based culture medium (1) .
(1) Means followed by equal letters within a cultivar do not differ by the Tukey's test, at 5% probability.
Table 3. Basal medium -Murashige and Skoog (MS), Murashige and Tucker (MT), and woody plant medium (WPM) -influence on shoot organogenesis from mature internodal segments of four citrus cultivars, after 60 days of in vitro culture (1) .
(1) Means followed by equal letters within a cultivar do not differ by the Tukey's test, at 5% probability.concentration of nitrogen and potassium in WPM could account for the differences in shoot size (Kobayashi et al., 2003).
Bacterial contamination during in vitro culture of mature internodal segments from greenhouse-grown mother plants is usually responsible for the loss of explants cultured on antibiotic-free medium.However, addition of antibiotics to the culture medium requires a careful evaluation of their effects on plant regeneration, since several reports have shown that they could have positive or negative effects on in vitro morphogenesis (Costa et al., 2000;Tang et al., 2004;Mendes et al., 2009).
Buds from mature explants, irrespective of the cultivar, were significantly influenced by the type and concentration of the antibiotics.Among the evaluated beta-lactams, the best responses of the mature explants were obtained with 500 mg L -1 cefotaxime, since it either promoted ('Pêra', 'Valência' and 'Bahia') or had no effect on shoot regeneration ('Cravo') (Table 2).Buds and shoots did not show any kind of burning.In 'Pêra', the mean number of shoots per explant increased from 2.5 (Table 2) to 4.03 (Table 4).In 'Valência' and 'Bahia', the same concentration of cefotaxime also increased shoot formation from 54.5 and 65.44% (Table 2) to 83.62 and 76.24% (Table 4), respectively.All other antibiotics had negative effects on shoot regeneration, except for timentin at 500 mg L -1 and augmentin at 500 mg L -1 , which did not inhibit shoot regeneration in 'Pêra', 'Valência' or 'Bahia'.The lowest concentrations of beta-lactam antibiotics (25 mg L -1 meropenem, 300 mg L -1 timentin and 250 mg L -1 augmentin) were not able to suppress bacterial growth.In explants cultured on 250 mg L -1 augmentin, bacteria growth was observed within seven days of culture.
These observations were quite surprising, since it has been recently reported that, in contrast to meropenem and timentin, cefotaxime caused a negative effect on shoot regeneration from epicotyl explants of citrus (Mendes et al., 2009).It has also been recently reported that meropenem was more effective against Agrobacterium and also improved transformation efficiencies in tobacco, tomato and rice, in comparison to cefotaxime and carbenicillin (Ogawa & Mii, 2004, 2005, 2007).
3. None of beta-lactams antibiotics tested is able to completely suppress bacterial contamination; however, the use of 500 mg L -1 cefotaxime in the culture medium is advantageous both to control bacterial contamination and to influence positively the morphogenetic process, especially in sweet orange.

Table 1 .
Factorial analysis of variance for the percentage of explants forming shoots and average number of shoots per explant, in internodal segments of Citrus sinensis and C. limonia cultured for 60 days in different plant growth regulators concentrations (PGRC), basal culture media (BCM) or antibiotic concentrations (AC).